Restriction endonucleases are a class of enzymes that occur naturally in prokaryotic and eukaryotic organisms. When restriction endonucleases are purified away from other contaminating cellular components, the enzymes can be used in the laboratory to cleave DNA molecules in a specific and predictable manner. Thus, restriction endonucleases have proved to be indispensable tools in modern genetic research.
Restriction endonucleases cleave DNA by recognizing and binding to particular sequences of nucleotides (the "recognition sequence") along the DNA molecule. The enzymes cleave both strands of the DNA molecule within, or to one side of, this recognition sequence.
Different restriction endonucleases have affinity for different recognition sequences. About 100 kinds of different endonucleases have so far been isolated from many microorganisms, each being identified by the specific base sequence it recognizes and by the cleavage pattern it exhibits. In addition, a number of restriction endonucleases, called restriction endonuclease isoschizomers, have been isolated from different microorganisms which in fact recognize the same recognition sequence as those restriction endonucleases that have previously been identified. These isoschizomers, however, may or may not cleave the same phosphodiester bond as the previously identified endonuclease.
Modification methylases are complementary to their corresponding restriction endonucleases in that they recognize and bind to the same recognition sequence. However, in contrast to restriction endonucleases, the modification methylases chemically modify certain nucleotides within the recognition sequence by the addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. Thus, in nature, methylases serve a protective function, i.e., to protect the DNA of an organism which produces its corresponding restriction enzyme.
Restriction enzymes and modification methylases can be purified from the host organism by growing large amounts of cells, lysing the cell walls, and purifying the specific enzyme away from the other host proteins by extensive column chromatography. However, the amount of restriction enzyme relative to that of the other host proteins is usually quite small. Thus, the purification of large quantities of restriction enzymes or methylases by this method is labor intensive, inefficient, and uneconomical.
An alternative method for producing large quantities of restriction and modification enzymes is to clone the genes encoding the desired enzymes and overexpress the enzymes in a well studied organism, such as Escherichia coli(E. coli). In this way, the amount of restriction and modification enzymes, relative to that of the host proteins, may be increased substantially. The first cloning of a DNA endonuclease gene was described by Mann et al. Gene 3:97-112 (1978). Since then more than seventy DNA methylase and restriction endonucleases have been cloned. Thus far, the majority of the restriction endonuclease genes are closely linked to their corresponding methylase genes.
Restriction-modification systems can be cloned by several methods. A number of endonuclease and methylase genes have been cloned from endogenous plasmids: EcoRII (Kosykh et al., Mol. Gen. Genet. 178:717-718 (1980)), EcoRI (Newman et al., J. Biol. Chem. 256:2131-2139 (1981)), Greene et al., J. Biol. Chem. 256:2143-2153 (1981)), EcoRV (Bougueleret et al., Nucl. Acids Res. 12:3659-3676 (1984)), PvuII (Blumenthal et al., J. Bacteriol. 164:501-509 (1985)), and PaeR71 (Gingeras et al., Proc. Natl. Acad. Sci. USA 80:402-406 (1983)). An alternative method of cloning is the phage restriction method in which bacterial cells carrying cloned restriction and modification genes survive phage infection (Mann et al., supra; Walder et al., Proc. Natl. Acad. Sci. U.S.A. 78:1503-1507 (1981); Rodicio et at., Mol. Gen. Genet. 213:346-353 (1988)). Another procedure is based upon methylation protection and has been suggested by Mann et at., supra, and Szomolanyi et al., Gene 10:219-225 (1980 ). This latter scheme involves digestion of a plasmid library with the restriction enzyme to be cloned. Only those plasmids with DNA sequences modified by the corresponding methylase will be resistant to digestion and will produce transformants in a suitable host. This selection method has been used to clone endonuclease and methylase genes together as well as to clone methylase genes alone (Szomolanyi et at., supra; Janulaitis et al., Gene 20:197-204 (1982); Walder et al., J. Biol. Chem. 258:1235-1241 (1983); Kiss et al., Gene 2l:111-119 (1983); Wilson, Gene 74:281-289 (1988)). However, this technique sometimes yields only the methylase gene, even though the endonuclease and modifying genes are closely linked.
A multi-step approach has been required to clone certain restriction-modification systems in E. coli, including DdeI (Howard et al., Nucl. Acids Res. 14:7939-7950 (1989)), BamHI (Brooks et al., Nucl. Acids Res. 17:979-997 (1989)), and KpnI (Hammond et al., supra). In each case, protection of the host with methylase expressed on a plasmid was necessary to stabilize a compatible vector containing the functional endonuclease gene. Wilson, supra, has proposed a model to explain why certain restriction-modification systems must be cloned utilizing a protected host. This model proposes that in order to establish a plasmid carrying a restriction-modification system, methylase protection must occur at a rate that is greater than the rate of endonuclease digestion. Otherwise, restriction enzymes would cleave unmethylated plasmid and/or genomic DNA and degrade the plasmid and/or kill the host. Although this model is a plausible explanation of plasmid establishment, it has yet to be determined whether continued independent expression of methylase from a separate plasmid is necessary to maintain the plasmid carrying the restriction-modification system during cell growth and replication.